In my last funded VA merit I proposed to investigate novel molecular mechanisms by which angiotensin II (Ang II), aldosterone and a WD (high in saturated fat and refined carbohydrates individually and collectively promotes insulin (INS) resistance in cardiovascular (CV) and skeletal muscle tissue in male rodents. Because of the increasing awareness by the NIH, the VA, the American Diabetes Association and American Heart Association regarding incorporation of females in research, we performed comparable work in females. We have found that this diet has a more negative CV impact in females. This is of translational relevance to the VA because of increasing numbers of female veterans and because in conditions of INS resistance such as obesity and type 2 diabetes, women display a substantially increased risk for CVD. As the lifetime risk for overweight/obesity and diabetes in women is high, associated CVD in women has become a major health problem. As people become obese and INS resistant, they manifest increasing CV stiffness, an abnormality that tracks closely with increasing CVD. INS resistance in the heart and vasculature results in decreased bioavailable nitric oxide (NO) which is associated with increased CV stiffness. Reduced bioavailable NO results in increased activity of the enzyme transglutaminase 2 (TG2), which increases collagen crosslinking and associated heart and vascular stiffness. We have observed that females, but not males, develop CV stiffness after only 8 weeks of consumption of a WD. Our ongoing work in a female mouse model of INS resistance induced by a WD also demonstrates that mineralocorticoid receptor (MR) blockade improves heart and vascular INS resistance and stiffness. We have garnered evidence that selective knockout of the endothelial cell (EC) MR in female mice abrogates the reduction in CV INS metabolic signaling and CV stiffness and impaired relaxation induced by consumption of a WD for 16 weeks. The role of the ECMR in the genesis of sex-related differences in CV INS signaling and stiffness over time has not been explored. In this proposal, our central hypothesis is that ECMR activation promotes CV INS resistance and stiffness. The corollary to this hypothesis is that impairment in INS metabolic signaling reduces bioavailable NO, which results in extracellular release and activation of TG2 promoting collagen crosslinking and therefore CV stiffness in females and males. In this revised proposal we plan to use a novel rodent model of endothelial specific MR knockout mice fed a WD, as well as innovative techniques to access INS resistance and associated CV stiffness in vivo and ex vivo. In Objective 1, we will determine the role of ECMR-mediated EC stiffening and resultant relationship between impairment of INS metabolic signaling and CV fibrosis/stiffness and impaired relaxation in males and females consuming a WD. relationship between ECMR-mediated impairment of INS metabolic signaling and reduced bioavailable NO and CV stiffness in WD- fed mice. In Objective 2, we will determine the role of ECMR mediated EnNaC activation in promotion of CV INS resistance in relation to vascular and cardiac fibrosis/stiffness and impaired relaxation. We anticipate that results from this proposal will yield unique insights into the mechanisms of CVD in obese and type 2 diabetic Veteran men and women, with the goal of translating these findings into therapeutic strategies to reduce CVD, especially in overweight INS resistant men and women.